Abstract

Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression.

Dynamics of Rb SUMOylation and phosphorylation during the cell cycle. (A) Rb is SUMOylated at early G1 phase. HEK293 cells were synchronized at the G0, early G1, G1, S and G2/M phases of the cell cycle, as described in the Materials and Methods section. The lysates in RIPA buffer were subjected to SDS-PAGE or immunoprecipitation using anti-Rb antibody and then blotted with SUMO1 antibody. (B) Rb is gradually phosphorylated after late G1 phase. HEK293 cells were synchronized and lysed as described above, followed by Western blot analysis with phosphorylated-Rb (Ser 807/811) antibody. Quantification of the data are represented as the mean ± the SEM (n = 3).

SUMOylation of Rb promotes its phosphorylation. (A) Diagram of the Ubc9 fusion-directed SUMOylation (UFDS) constructs of Rb. (B) Constitutive SUMOylation of Rb caused by UFDS. HEK293 cells transiently transfected with His-tagged UFDS constructs were lysed in RIPA buffer with or without NEM, and then blotted with anti-His antibody. Rb-Ubc9: unmodified Rb-Ubc9; Rb-Ubc9-S: SUMOylated Rb-Ubc9. (C) UFDS of VCP promotes its phosphorylation level. HEK293 cells were co-transfected with indicated constructs. Lysates were incubated with Ni-NTA agarose beads to pull down His-tagged Rb-Ubc9 or Ubc9 (used as negative control), and then immunoblotted with anti-SUMO1, anti-phosphorylated-Rb (Ser 807/811) or anti-His antibodies. Hyper-pRb-Ubc9: hyper-phosphorylated Rb-Ubc9. The relative phosphorylation level of each form of the Rb-Ubc9 fusion proteins were quantified and represented as the mean ± the SEM(n = 4). (D) A mild increase in global SUMO-1 conjugation is sufficient to enhance Rb phosphorylation. The phosphorylation levels of endogenous Rb in HEK293 cells expressing increasing amounts of GFP-SUMO-1 were determined as above. The results represent the mean± the SEM (n = 3). (E) The SUMO-deficient K720R mutation reduces the SUMOylation and phosphorylation of Rb. HEK293 cells were co-transfected with the WT or mutant Rb-His constructs together with SUMO-related plasmids, and analyzed for SUMO conjugation and phosphorylation as described above.

CDK2 is required for SUMOylation-enhanced phosphorylation of Rb. (A) UFDS of Rb stimulates its binding with CDK2. HEK293 cells were co-transfected with His-tagged Rb-Ubc9 together with Flag-tagged CDKs. The binding capability of Rb-Ubc9 with each CDK was analyzed by pull down assay 72 h post-transfection. Data are shown as the mean± the SEM (n = 3). (B, C) Improved binding of Rb to endogenous CDK2 caused by UFDS (B) or enhanced of global SUMOylation (C). HEK293 cells were transfected as indicated. The binding of Rb to CDK2 was determined as described above. These results are represented as the mean± the SEM (n = 3). PD: pull down. (D) The SUMO-deficient K720R mutation exhibited reduced binding to CDK2. HEK293 cells were co-transfected with the WT or mutant Rb-His constructs together with GFP-SUMO1, and analyzed for CDK2 binding as described above. PD: pull down. (E) Inhibition of CDK2 by siRNA blocked the SUMOylation-enhanced Rb phosphorylation. HEK293 cells were first transfected with GFP-SUMO1, and they were then transfected with control or CDK2 siRNAas indicated. Rb phosphorylation was determined using an antibody against Rb S807/S811 (n = 4 independent experiments), and the values represent the mean± the SEM.

The recruitment of CDK2 to SUMOylated Rb is mediated by a functional SIM. (A) The alignment of the sequences corresponding to the putative CDK2 SIM in various species with the consensus SIM site. x stands for any amino acid. (B) CDK2 binds non-covalently to SUMO1 in vitro. Recombinant GST-tagged SUMO1 were incubated with His-tagged CDK2 and control GFP, followed by affinity pull down with Ni-NTA beads and Western blot. PD: Pull down. (C) The CDK2 SIM is required for its enhanced recruitment to SUMOylated Rb. HEK293 cells were co-transfected with His-tagged Rb-Ubc9 together with Flag-tagged wild-type CDK2 and a mutant lacking the SIM (ΔSIM) for the binding capacity assay as described in .

SUMOylation of Rb disrupts the E2F1-Rb interaction. (A) The constitutive SUMOylated Rb construct shows reduced interaction with E2F1. HEK293 cells were transfected with His-tagged Rb-Ubc9 or Ubc9, followed by pull-down experiments. The amount of Rb-bound E2F1 was determined by immunoblotting. Quantification of the data is shown as the mean± the SEM (n = 4). (B) Elevated global SUMOylation causes decreased association between Rb and E2F1. HEK293 cells transfected as indicated were lysed and subjected to pull down assay. Rb and E2F1 were detected by Western blot protein gel blot analysis using the indicated antibodies. The mean (n = 3) with the SEM values for the amount of Rb-bound E2F1 are shown. PD: Pull down. (C) Defective SUMOylation of Rb leads to increased sequestration of E2F1. WT and K720 Rb were precipitated by Ni-NTA using lysates from HEK293 cells transfected with the indicated constructs, and the amount of associated E2F1 was determined by immunoblotting. Data are expressed as the mean ± the SEM (n = 3). PD: pull down.